The present invention is in the field of biotechnology and relates to recombinant DNA techniques, in particular to the production of synthetic peptides coding for pre-M/M protein of Dengue virus serotype 2 and chimeric proteins which contain epitopes of pre-M/M protein of Dengue virus serotype 2 and 4.
The technical objective is to identify Pre-M/M neutralizing and protective epitopes, cross reactive for all dengue virus serotypes to obtain an immunogen for human vaccination.
Dengue virus belongs to the Flavivirus genus, family Flaviviridae (Westaway, E. G. et al. 1985. Flaviviridae. Intervirol. 24 p.183). It is an enveloped virus with a single RNA chain of positive polarity as genetic material, which codes for a polyprotein processed co- and post-transductionally by cellular and viral proteases.
There are two structural proteins in the viral membrane: E (envelope) and M (membrane), while there are several copies of the other structural protein, C (capside) forming the isometric nucleocapside. Besides, at least seven non-structural proteins have been identified (NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5).
Glycoproteins E and NS1 are individually able to offer active and passive protection against the homologous serotype of Dengue virus, while the highly conformational complexity of the relevant epitopes is preserved. For this reason, recombinant eukaryotic cellular systems have been mainly selected for the immunological evaluation of these proteins, for example vaccinia virus (Bray, M. et al. 1989. Mice immunized with recombinant Vaccinia virus expressing dengue-4 structural proteins with or without nonstructural protein NS1 are protected against fatal dengue virus encephalitis. J. Virol. 63 p.2853) and baculovirus (Zhang, Y. M. et al. 1988. Immunization of mice with dengue structural proteins and nonstructural protein NS1 expressed by baculovirus recombinant induces resistance to dengue virus encephalitis. J. Virol. 62 p.3027).
The small protein M (8 kDa) is synthesized like a glycosylated precursor named pre-M (22 kDa approximately), which suffers a late endoproteolitic cleavage just before or after the liberation of the virus of the infected cell (Murray, J. M. et al. 1993. Processing of the dengue virus type 2 proteins prM and C-prM. J. Gen. Virol. 74 p.175). The cleavage, which is probably done by a cellular protease, seems to happen in the post-Golgi acidic vesicles, being inhibited by agents that destabilized the low pH of this vesicles (Randolph, V. B. et al. 1990. Adidotropic amines inhibit proteolytic processing of Flavivirus prM protein. Virol. 174 p.450). The fragment pre- has been identified in vitro only in the extracellular medium, its destiny in vivo remains unknown (Murray, J. M. et al. 1993. Processing of the dengue virus type 2 proteins prM and C-prM. J. Gen. Virol. 74 p.175).
It is thought that the function of pre-M/M during the Flavivirus exocytic via is to avoid the activation of the fusogenic membrane domain of E with the acidic pH of the environment (Randolph, V. B. et al. 1990. Acidotropic amines inhibit proteolytic processing of Flavivirus prM protein. Virol. 174 p.450); if this event happens, then the viral liberation will be prevented. In fact, it has been determined that pre-M and E interact in the immature intracellular virions (Wengler, G. y Wengler, G. 1989. Cell-associated West Nile flavivirus is covered with E+pre-M protein heterodimers which are destroyed and reorganized by proteolytic cleavage during virus release. J. Virol. 63 p.2521), and that the native conformation of E it is only acquired in the presence of pre-M (Konishi, E. y Mason, P. W. 1993. Proper maturation of the Japanese encephalitis virus envelope glycoprotein requires cosynthesis with the premembrane protein. J. Virol. 67 p.1672). In addition, already liberated virions that only have pre-M in their membranes show, in general, a lower infectivity than the completely mature virion (Wengler, G. y Wengler, G. 1989. Cell-associated West Nile flavivirus is covered with E+pre-M protein heterodimers which are destroyed and reorganized by proteolytic cleavage during virus release. J. Virol. 63 p.2521), in which although M and pre-M are present, the former is predominant.
Pre-M and M offer an active protection when they have been expressed in recombinant vaccinia virus, but this do not happen with the fragment pre- (Bray, M. y Lai, C.-J. 1991. Dengue virus premembrane and membrane proteins elicit a protective immune response. Virol. 185 p.505), besides the combination pre-M or M with glycoprotein E in the same recombinant Vaccinia virus gives in general levels of protection higher than those reached by each protein individually. Similarly, certain antibodies against pre-M/M are able to protect passively in mice (Kaufman, B. M. et al. 1989. Monoclonal antibodies for dengue virus prM glycoprotein protect mice against lethal dengue infection. Am J. Trop. Med. and Hyg. 41 p.576).
The use of synthetic peptides has allowed to establish the molecular basis of antigenicity according to spacial conformation and the immunological properties of the antigen involved [Arnon, R. y Sela, M. 1985. Synthetic Vaccines: present and future. Ann. Inst. Pasteur/Immunol 136 D, 271-282]. The synthetic peptides as anti-dengue vaccine subunits will allow to include in the final formulation only the protective epitopes that do not cause immune-amplification (Halstead, S. B., y O""Ruourke, E. J. 1977. Dengue viruses and mononuclear phagocytes. I. Infection enhancement by non-neutralizing antibody. J. Exp. Med. 146 p.201; Halstead, S. B. 1979. In vivo enhancement of dengue virus infection in rhesus monkeys by passively transferred antibody. J. Infect. Dis. 140 p.527), or altematively, to include protective peptides of each of the four serotypes. The characterization of the antigenic determinants of E and NS1 has been carried out successfully. However, there are no similar studies on the also important protein pre-M/M, that is why the results of this paper are a first step on that direction.
The efforts to express the flaviviral protein pre M, M and E in E. coli have not always been successful (Chambers, T. J. et al. 1990. Production of yellow fever virus proteins in infected cells: identification of discrete polyprotein species and analysis of cleavage kinetics using region-specific polyclonal antiserum. Virol. 177 p.159; Yan, B.-S. et al. 1994. Truncating the putative membrane association region circumvents the difficulty of expressing hepatitis C virus protein E1 in Escherichia coli. J. Virol. Meths. 49 p.343). Apparently, the hydrophobic regions these protein have in C-terminal are the cause of the low or undetectable heterologous expression levels (Yan, B.-S. et al. 1994. Truncating the putative membrane association region circumvents the difficulty of expressing hepatitis C virus protein E1 in Escherichia coli. J. Virol. Meths. 49 p.343).
The expression of those proteins (as well as NS1) in E.coli, in general have been obtained by fusion (fragmented or not) to other bacterial proteins (e.g. b -galactosidase (Cane, P. A. y Gould, E. A. 1988. Reduction of yellow fever mouse neurovirulence by immunization with a bacterially synthesized non-structural protein (NS1) fragment. J. Gen. Virol. 69 p.1241), TRPE (Megret, F. et al. 1992. Use of recombinant fusion proteins and monoclonal antibodies to define linear and discontinuous antigenic sites on the Dengue envelope glycoprotein. Virol. 187 p.480) and the protein A of Staphylococcus aureus (Murray, J. M. et al. 1993. Processing of the dengue virus type 2 proteins prM and C-prM. J. Gen. Virol. 74 p.175). In these fusion proteins most of the relevant conformational epitopes are absent, because though the antisera generated against them can recognize the whole virus, they are not able neither to neutralize it nor to inhibit their hemagglutinating properties (Megret, F. et al. 1992. Use of recombinant fusion proteins and monoclonal antibodies to define linear and discontinuous antigenic sites on the Dengue envelope glycoprotein. Virol. 187 p.480). However, recent reports show that the solubility of the fusion proteins, and as a consequence, the use of non-denaturalizing methods for its purification, may preserve most of the neutralizing (Seif, S. A. et al. 1995. Finer mapping of neutralizing epitope(s) on the C-terminal of Japanese encephalitis virus E-protein expressed in recombinant Escherichia coli system. Vaccine 13 p.1515) and protective (Srivastava, A. K. et al. 1995. Mice immunized with a dengue type 2 virus E and NS1 fusion protein made in Escherichia coli are protected against lethal dengue virus infection. Vaccine 13 p.1251) epitopes they possess.
In the case of pre-M/M, its pre-domain has 6 cysteines involved in 3 disulfide bridges, as well as an N-glycosylation site in the asparagine 69. The structure of E and NS1 is even more complicated; it involves 6 disulfide bridges and several N-glycosylation sites. However, the little ectodomain of M is apparently free of those conformational complexities because it does not have cysteines, and it is not glycosylated in its natural form.
The insertion of heterologous fragments in permissive areas of immunogenic proteins which topology is more or less known and immunization of these fusions is a complementary alternative to the use of synthetic peptides. Both strategies allow defining the presence of sequential B cell, as well as T cell epitopes. The biological importance of these epitopes could be experimentally evaluated to decide where to include them or not in certain vaccine preparation.
The invention, provides synthetic peptides of Dengue virus preM/M protein comprising amino acids 3-11 (Sequence ID No.:1), 45-67 (Sequence ID No.:2), 57-92 (Sequence ID No.:3) 69-93 (Sequence ID No.:4) and 103-124 (Sequence ID No.:5) that include at least one opitope that is cross reactive with any Dengue virus serotype; and mimetic compounds thereof.
Also provided are diagnostic tests and pharmaceutical formulations that include the above peptides or mimetic compounds useful in the detection and prophylaxis or treatment of flavivirus infections.
The invention further provides antibodies and antibody fragments that specifically bind the Dengue virus preM/M protein comprising amino acids 3-31 (Sequence ID No.:1), 45-67 (Sequence ID No.:2), 57-92 (Sequence ID No.:3), 69-93 (Sequence ID No.:4) and 103-124 (Sequence ID No.:5) that include at least one epitope that is cross reactive with any Dengue virus scrotype. Vaccine and therapeutic preparations that include these antibodies are also provided.
Further, the invention provides genetic constructs that include amino acid sequences that include epitopes of pre-M/M from Dengue virus scrotypes 2 and 4, fused to a carrier protein.
Five peptides from Pre-M/M protein of Dengue 2 virus which cover 58% of the aminoacid sequence (97/166 AA) were chemically synthesized. They were 3-31; 45-67; 57-92; 69-93; and 103-124, which were subsequently named B 19-6; B 20-2; B 19-5; B 20-1; B 20-3 respectively.
Peptides were inoculated in Balb/c mice both conjugated or not to a carrier protein. The sera obtained after immunization with the conjugated peptides were tested by in vitro neutralization by reducing the number of plaques and by ELISA. We also studied the active protection against a Dengue 2 viral challenge in the immunized mice.
In the case of mice immunized with the non-conjugated peptides, the antibody response was evaluated by ELISA and the proliferative response of spleen T lymphocytes against Dengue 2 virus was evaluated too. Fusion proteins were also obtained, and two of the four regions covered by peptides (1-42 and 92-133) were inserted to them and were expressed in E.coli bacteria. Immunization with these fusions will complement the results obtained with the synthetic peptides.
The presence of B cell epitopes in both mice and humans was demonstrated as the peptides were recognized by antibodies from the immunized mice and by sera from patients who had the clinical and serological diagnosis of Dengue virus, using ELISA in both cases. Peptides 19-6 and 20-3 were able to induce neutralizing antibody production against the four Dengue virus serotypes.
Virus-specific proliferative responses were demonstrated in mice immunized with non-conjugated peptides 19-6 and 19-5. Mice immunized with conjugated peptides 19-6, 20-1, and 19-5 showed a statistically significative level of protection when they were challenged with Dengue 2 virus.
Thus, the presence of sequential epitopes in Pre-M/M protein of Dengue virus 2 was demonstrated, as well as their relevance in the immune response against these flaviviruses.